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      GDF5 Mediated Enhancement of Chondrocyte Phenotype and its Modulation by Heparin and Heparan Sulfates

      Ayerst, Bethanie Imogen

      [Thesis]. Manchester, UK: The University of Manchester; 2017.

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      Abstract

      Articular cartilage plays a vital role in load-bearing joints, providing an almost frictionless surface to articulating bones. However, the avascular nature and low cell density of the tissue means that following injury, there is limited potential for regeneration and repair. With the ageing population, the prevalence and economic burden associated with osteoarthritis (OA) is increasing rapidly, but as of yet there are no fully effective ways to treat the condition. Research into novel therapies has therefore become a popular avenue of investigation, and human mesenchymal stem/stromal cells (hMSCs) have been highlighted as particularly promising targets. However, current, methods for inducing the chondrogenic differentiation of hMSCs, which typically employ the use of transforming growth factor beta 1 or 3 (TGFβ1/3), result in the production of hypertrophic rather than hyaline tissue, hampering translational progress. Growth differentiation factor 5 (GDF5) belongs to the TGFβ superfamily of proteins and is vital for skeletal formation, however its use in cartilage tissue engineering (TE) strategies has been somewhat neglected. Here we demonstrate that GDF5 significantly increases aggrecan gene expression (a marker of articular cartilage), without affecting collagen type X expression (a marker of chondrocyte hypertrophy), in chondrocyte pellet cultures derived from hMSCs, making it a promising target for the formation of permanent articular cartilage.The therapeutic application of growth factors is, at present, limited due to their expense, susceptibility to proteolytic degradation, and rapid clearance, leading to large quantities being required to get anywhere near the desired outcome. The highly sulfated glycosaminoglycan (GAG), heparin, is already extensively used in the clinic as an anticoagulant, and is also able to bind and potentiate the activity of a wide range of growth factors. As such, researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery/ activity of growth factors in TE strategies. Here, we identify GDF5 as a novel heparin/heparan sulfate (HS)-binding protein, and show that endogenous HS proteoglycans (HSPGs) are vital for localizing GDF5 to the cell surface, but are not required for its signalling activity. Importantly, we report that clinically relevant doses of heparin (≥ 10 nM), but not equivalent concentrations of HS, inhibit GDF5’s biological activity, in both hMSC-derived chondrocyte pellet cultures, and in the skeletal cell line ATDC5. We demonstrate that these inhibitory effects are due to heparin (but not HS) inhibiting both GDF5 binding to endogenous HSPGs and GDF5-induced induction of Smad 1/5/8 signalling. This study may therefore explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE, and the adverse skeletal effects, such as osteoporosis, that have been reported in the clinic following long-term heparin treatment. Together, our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g. HS derivatives or synthetics), with better-defined structures and fewer off-target effects, if optimal therapy is to be achieved. In the case of GDF5, we have used a variety of developed techniques to begin uncovering important structural and functional information regarding the HS-GDF5 interaction, which are hoped to ultimately pave the way towards achieving this aim. Although further analysis is necessary, our data indicate that relatively long HS sequences are required for binding, and that both ionic and non-ionic interactions play a role in the interaction. In addition we suggest that low- rather than high-affinity HS variants may be key to potentiating the activity of this growth factor.

      Bibliographic metadata

      Type of resource:
      Content type:
      Administered thesis
      Form of thesis:
      Traditional
      Type of submission:
      Doctoral level ETD - final
      Thesis title:
      GDF5 Mediated Enhancement of Chondrocyte Phenotype and its Modulation by Heparin and Heparan Sulfates
      Degree type:
      Doctor of Philosophy
      Degree programme:
      PhD Singapore A*STAR 4yr (CMB)
      Publication date:
      2017-01-21T11:06:35
      Institution:
      The University of Manchester
      Location:
      Manchester, UK
      Total pages:
      175
      Abstract:
      Articular cartilage plays a vital role in load-bearing joints, providing an almost frictionless surface to articulating bones. However, the avascular nature and low cell density of the tissue means that following injury, there is limited potential for regeneration and repair. With the ageing population, the prevalence and economic burden associated with osteoarthritis (OA) is increasing rapidly, but as of yet there are no fully effective ways to treat the condition. Research into novel therapies has therefore become a popular avenue of investigation, and human mesenchymal stem/stromal cells (hMSCs) have been highlighted as particularly promising targets. However, current, methods for inducing the chondrogenic differentiation of hMSCs, which typically employ the use of transforming growth factor beta 1 or 3 (TGFβ1/3), result in the production of hypertrophic rather than hyaline tissue, hampering translational progress. Growth differentiation factor 5 (GDF5) belongs to the TGFβ superfamily of proteins and is vital for skeletal formation, however its use in cartilage tissue engineering (TE) strategies has been somewhat neglected. Here we demonstrate that GDF5 significantly increases aggrecan gene expression (a marker of articular cartilage), without affecting collagen type X expression (a marker of chondrocyte hypertrophy), in chondrocyte pellet cultures derived from hMSCs, making it a promising target for the formation of permanent articular cartilage.The therapeutic application of growth factors is, at present, limited due to their expense, susceptibility to proteolytic degradation, and rapid clearance, leading to large quantities being required to get anywhere near the desired outcome. The highly sulfated glycosaminoglycan (GAG), heparin, is already extensively used in the clinic as an anticoagulant, and is also able to bind and potentiate the activity of a wide range of growth factors. As such, researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery/ activity of growth factors in TE strategies. Here, we identify GDF5 as a novel heparin/heparan sulfate (HS)-binding protein, and show that endogenous HS proteoglycans (HSPGs) are vital for localizing GDF5 to the cell surface, but are not required for its signalling activity. Importantly, we report that clinically relevant doses of heparin (≥ 10 nM), but not equivalent concentrations of HS, inhibit GDF5’s biological activity, in both hMSC-derived chondrocyte pellet cultures, and in the skeletal cell line ATDC5. We demonstrate that these inhibitory effects are due to heparin (but not HS) inhibiting both GDF5 binding to endogenous HSPGs and GDF5-induced induction of Smad 1/5/8 signalling. This study may therefore explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE, and the adverse skeletal effects, such as osteoporosis, that have been reported in the clinic following long-term heparin treatment. Together, our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g. HS derivatives or synthetics), with better-defined structures and fewer off-target effects, if optimal therapy is to be achieved. In the case of GDF5, we have used a variety of developed techniques to begin uncovering important structural and functional information regarding the HS-GDF5 interaction, which are hoped to ultimately pave the way towards achieving this aim. Although further analysis is necessary, our data indicate that relatively long HS sequences are required for binding, and that both ionic and non-ionic interactions play a role in the interaction. In addition we suggest that low- rather than high-affinity HS variants may be key to potentiating the activity of this growth factor.
      Keyword(s):
      Thesis main supervisor(s):
      Thesis co-supervisor(s):
      Degree grantor:
      Language:
      en

      Institutional metadata

      University researcher(s):
      Academic department(s):

        Record metadata

        Manchester eScholar ID:
        uk-ac-man-scw:307032
        Created by:
        Ayerst, Bethanie
        Created:
        21st January, 2017, 11:06:35
        Last modified by:
        Ayerst, Bethanie
        Last modified:
        3rd February, 2017, 10:54:33

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